Project Summary Antibiotic resistant bacterial pathogens are a major threat to public health. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen displaying high levels of antibiotic resistance even in the absence of novel acquired mutations. This resistance is mediated in part by the outer membrane barrier, which also contributes to resistance to host-derived antimicrobial peptides and is an essential structure for bacterial survival. Despite this, relatively few inhibitors targeting outer membrane biogenesis have been developed and multiple elements of outer membrane biology are poorly understood. This project has used Insertion Sequencing (InSeq) as a transposon mutagenesis strategy to identify genetic determinants of envelope- mediated intrinsic antibiotic resistance. Informed by InSeq results we used a novel resource, an arrayed library of P. aeruginosa strains expressing camelid intrabodies, to identify candidate inhibitors of P. aeruginosa outer membrane barrier function. This proposed work will explore the phenotypic impacts of the 14 most promising candidate inhibitors and identify binding partners of five selected intrabodies in order to propose mechanisms of action and gain new insights into major antibiotic resistance mechanisms in this opportunistic pathogen.